The present invention relates generally to chelating agents for the binding of metal ions to biologically active molecules, both naturally occurring and synthetic. Specifically, it relates to bifunctional chelating agents comprising an array of metal binding groups plus a single additional moiety (hereafter a "substrate reactive" group) which is reactive with one or more functionalities present on the molecule to be labelled with a metal ion (hereafter the "substrate"). According to one embodiment, the invention relates to antibody conjugates and antibody metal ion conjugates comprising the bifunctional chelating agents and to the use of such antibody-metal ion conjugates for in vivo diagnostic imaging methods utilizing radiation emitting and radiation absorbing metal ions. The invention additionally relates to therapeutic methods involving the use of such antibody-metal ion conjugates which emit cytotoxic radiation.
Of interest to the present invention are disclosures showing the use of protein/metal ion conjugates for diagnostic and therapeutic purposes. Gansow, et al., U.S. Pat. No. 4,454,106 discloses the use of monoclonal antibody/metal ion conjugates for in vivo and in vitro radioimaging diagnostic methods. Goldenberg, et al., N. Eng. J. Med., 298 1384-88 (1978) discloses diagnostic imaging experiments wherein antibodies to the known tumor associated antigen carcinoembryonic antigen (CEA) are labelled with iodine.sup.131 and injected into patients with cancer. After 48 hours, the patients are scanned with a gamma scintillation camera and tumors are localized by the gamma emission pattern.
Other workers disclose the therapeutic use of antibody/metal ion conjugates for delivery of cytotoxic radioisotopes to tumor deposits in vivo. Order, et al., Int. J. Radiation Oncology Biol. Phys., 12, 277-81 (1986) describes treatment of hepatocellular cancer with antiferritin polyclonal antibodies to which yttrium.sup.90 has been chelated. Duchsbaum, et al., Int. J. Radiation Oncology Biol. Phys., 12, 79-82 (1985) discloses radiolabelling of monoclonal antibodies to CEA with yttrium.sup.88 and suggests the possibility of localization and treatment of colorectal cancers therewith. Nicolotti, EPO Application No. 174,853 published Mar. 19, 1986, discloses conjugates comprising metal ions and antibody fragments. According to that disclosure, monoclonal antibodies of subclass IgG are enzymatically treated to remove the Fc fragment and reductively cleave the disulfide bond linking the antibody heavy chains. The Fab' fragment is then linked to a chelating agent bound to a radionuclide metal ion for in vivo diagnostic or therapeutic use.
Antibody/metal ion conjugates may be formed through the use of bifunctional chelating agents comprising an array of metal-binding groups plus a moiety capable of covalent binding to a protein substrate. Early work with bifunctional chelating agents involved the compound diethylenetriaminepentaacetic acid (DTPA) and its derivatives. This compound comprises a backbone of three nitrogen atoms linked by two ethylene chains. Extending from the nitrogen atoms on the backbone are five carboxymethyl moieties. Methods have been described whereby one of the carboxymethyl groups may be reacted to form an amide bond with an amino acid residue present on an antibody or other protein molecule. The other four carboxymethyl moieties, together with the three nitrogen atoms, then remain available for metal binding. Unfortunately, because there is no intrinsic difference between the substrate reactive and chelating functionalities in DTPA, such procedures can lead to cross linking and denaturation of the antibody with concomitant degradation of its ability to bind to the target antigen.
In order to avoid the potential for such undesired cross-linking, bifunctional chelating agents incorporating a unique protein substrate reactive site have been developed. The first such compounds were derivatives of the compound ethylenediaminetetraacetic acid (EDTA). This compound comprises a backbone of two nitrogen atoms linked by an ethylene chain. Extending from the nitrogen atoms on the backbone are four carboxymethyl moieties which with the nitrogen atoms are suitable for metal binding. The bifunctional chelating derivatives of EDTA are characterized by the attachment of a unique protein substrate reactive function at a methylene carbon of the polyamine backbone. Sundberg, et al., J. Med. Chem. 17, 1304 (1974) discloses the synthesis of an EDTA derivative bearing a para aminophenyl protein reactive substituent. This derivative may in turn be converted to bifunctional chelating agents capable of being coupled to protein substrates under mild conditions either by reaction of the amine with a portion of a chemically modified protein or by treatment of the primary amine to form other substituents capable of binding to protein substrates under mild conditions.
Meares, et al., J. Protein Chem., 3, 215-228 (1984) discloses methods whereby the para aminophenyl derivative is converted to a diazonium derivative through nitrous acid treatment, to an isothiocyanate derivated by treatment with thiophosgene, to a bromoacetamide derivative by treatment with bromoacetylbromide and to a palmitaamidobenzyl derivative by treatment with palmitoyl chloride. Altman, et. al., J. Chem. Soc. Perkin Trans. I., 365, 59-62 (1983) discloses a number of phenethyl analogues of the above EDTA compounds. See also, Sundberg, et al., U.S. Pat. No. 3,994,966.
Cyclic chelating agents are known in the art. Kroll, et al., Nature, 180 919-20 (1957) discloses the use of cyclohexane-1,2-trans-diaminetetraacetic acid for the removal of heavy metal ions from the human body. Moi, et al., Anal. Biochem., 148, 249-253 (1985) discloses a macrocyclic bifunctional chelating agent precursor named 6-(p-nitrobenzyl)-1,4,8,11-tetra-azacyclotetradecane N,N',N'', N'''-tetraacetic acid (p-nitrobenzyl-TETA) which forms a copper chelate which is extremely stable in human serum under physiological conditions. In addition, the p-bromoacetamidobenzyl derivative of TETA shows high stability after conjugation to a monoclonal antibody. The Moi, et al. reference also discloses that improved metal binding yields may be obtained in some cases where the conjugate contains a spacer group between the protein and TETA.
Of interest to the present application are the disclosures of Green, et al., Int. J. Nucl. Med. Biol., 12, 381-85 (1985) and Taliaferro, et al., Inorg. Chem. 23 1188-92 (1984) disclosing chelating agents. Green, et al. discloses a sexadentate ligand N,N'-dipyridoxylethylenediamine-N,N'-diacetic acid (PLED) complexed with gallium.sup.68 and indium.sup.111. Taliaferro, et al., discloses PLED chelates as well as those of N,N'-ethylene-bis[2-(o-hydroxy phenyl)glycine] (EHPG) and N,N'-bis(2- hydroxybenzyl) ethylenediamine ,N,N'-diacetic acid (HBED).
Other variations on known DTPA and EDTA derivatives include those of Brechbiel, et al., Inorg. Chem., 25, 2772-81 (1986) which discloses derivatives of DTPA wherein para-aminophenyl substituents are attached to the methylene carbons of the polyamine backbone. In addition, Altman, et al., J. Chem. Soc. Perkin Trans. I., 59 (1984) discloses a 2-carboxyethyl chelating derivative of EDTA.
Methods of synthesizing derivatives of DTPA and EDTA wherein the protein reactive functionality is attached to a methylene carbon of the polyamine backbone are complex, difficult to practice and have limited flexibility. In addition, the polyamine backbone carbon atoms of many chelating agents such as those wherein such carbons are part of a cyclic system are not readily available for substitution. It is therefore desired to develop bifunctional chelating agents having a unique substrate reactive function attached to a moiety common to, and accessible in, all polyaminopolycarboxylate frameworks. It is further desired to develop general methods for the synthesis of such compounds.
Of interest to the present invention is the disclosure of Takeshita and Maeda, Yukagaku, 19, 984-93 (1970) which shows a surfactant compound with the structure: ##STR1##
wherein R is a long chain alkyl.
Also of interest to the present invention is the disclosure of Borch, et al., J. Amer. Chem. Soc., 93 2397 (1971) showing the reductive amination of a variety of pyruvic acids including phenylpyruvic and para-hydroxyphenylpyruvic acid to the corresponding dl-alpha amino acids under mild conditions using ammonia and sodium cyanoborohydride.